EP2287332B1 - Nukleinsäuren und Verfahren zur Umweltüberwachung - Google Patents

Nukleinsäuren und Verfahren zur Umweltüberwachung Download PDF

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EP2287332B1
EP2287332B1 EP09010238.5A EP09010238A EP2287332B1 EP 2287332 B1 EP2287332 B1 EP 2287332B1 EP 09010238 A EP09010238 A EP 09010238A EP 2287332 B1 EP2287332 B1 EP 2287332B1
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seq
nucleic acid
acid molecule
dna
probe
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EP2287332A1 (de
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Kornelia Berghof
Astrid Grönewald
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Biotecon Diagnostics GmbH
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Biotecon Diagnostics GmbH
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/16—Primer sets for multiplex assays

Definitions

  • Environmental monitoring is an important part of the manufacture of sterile products, especially in the aseptic production of pharmaceuticals, but also for the production of non-sterile products.
  • the current good manufacturing practise cGMP
  • cGMP rigorous manufacturing practise
  • Taking samples for sterility testing is only significant for the whole production-lot if the microbiological status of the environment is tested at the same time.
  • These environmental tests have to be carried out regularly by monitoring the working area as well as the personnel and by using appropriate controls.
  • a functional environmental monitoring process is a prerequisite for the release of parenteral pharmaceuticals.
  • the requirements regarding the environmental monitoring depend on the risk that the environment would pose to the product quality.
  • the production facilities are therefore divided into defined critical and controlled areas with different demands on testing intervals and warning and action levels concerning total microbial counts.
  • Sampling methods vary for air, surfaces, and personnel, but all of them amount to the detection of microbial growth occurring on solid media (i.e ., agar plates) after an incubation period. If colonies are present on the plates, they are then further analyzed and identified at an appropriate level in order to determine the typical flora of the respective environment. Identification to a certain degree is also useful in order to draw conclusions as to the source of the contamination and to possible influence factors.
  • Identification of the organisms that grow on agar plates sampled in the process of environmental monitoring is usually performed with conventional phenotypical methods such as: (i) Gram stain, followed by (ii) microscopy to read the Gram stain reaction and determine the cellular morphology of the isolate, and depending on the results, (iii) biochemical tests. Throughout the different steps of theses conventional methods which in many cases can take days, secondary contaminations often occur. It is almost impossible to notice with these conventional methods when a bacterial colony is overgrown by another organism with similar characteristics.
  • Gram stain and microscopy are critical steps for the identification process as their results usually determine which further biochemical tests are conducted.
  • the outcome of a Gram stain can vary with the age of bacterial colonies, and with the quality of the reagents used for the test. Cell morphology may also vary with colony age, and even for the experienced microbiologist it is difficult to distinguish between short rods and large cocci under the microscope.
  • Biochemical tests usually rely on the direct or indirect detection of enzymes expressed by the microorganisms and therefore require freshly and densely grown pure cultures of single organisms. In stressed microorganisms ( e.g. , by temperature or selective growth media) the expression of the enzymes may be partly or fully inhibited. It is also well known to those experienced in the art that biochemical characteristics of a species only apply to the majority of isolates belonging to this species while there are always exceptional strains (Bergey 2001). Various studies have shown the limitations of commercial biochemical test systems concerning specificity (Brigante et al. 2008; Delmas et al. 2008).
  • Identification methods based on nucleic acid analyses include DNA-DNA hybridization, polymerase chain reaction (PCR), and sequencing.
  • sequencing of the 16S rDNA region has become a valuable tool in bacterial taxonomy and identification.
  • the generation of templates for sequencing, the sequencing reaction itself, and the analysis of the sequencing results is a time-consuming, expensive, and potentially error-prone process.
  • the present invention provides nucleic acids and methods for a rapid classification and identification of predetermined groups of bacteria typically occurring in the environment and on human skin and are therefore part of the baseline flora found in environmental monitoring.
  • Organisms not being a member of the predetermined groups are preferably not identified.
  • predetermined group is used to describe a part or the whole of the bacteria topically occurring in the environment and on human skin.
  • the members of the group can be chosen from one genus, but can also be chosen from different genera.
  • One preferred group consists of the genera Corynebacterium, Micrococcus, Kocuria, Kytococcus, and Nesterenkonla. Another preferable group consists of the genus Corynebactenum. Another preferable group consists of the species Micrococcus luteus.
  • the present invention provides inter alia a reliable and specific means for the detection of microorganisms of the taxonomic unit set forth by the genera Micrococcus, Kocuria, Kytococcus, Nesterenkonla, which optionally also allows for further determination and discrimination of other microorganisms of other taxonomic units and of one or more genera or species within the genera Micrococcus, Kocuria, Kytococcus, Nesterenkonla.
  • oligonucleotides or combinations thereof targeting the genomic region from the 3'-end of the 23S-rDNA to the 3'-end of the 5S-rDNA gene including the internal transcribed spacer region (ITS 2) enable such a specific detection of the typical environmental monitoring flora. They also discovered oligonucleotides targeting this region that allow a specific detection of the most frequent species found on environmental monitoring plates, i.e. Micrococcus luteus.
  • Figure 1 shows the DNA region as well as the hybridization positions of the oligonucleotides of this invention.
  • oligonucleotides involved identification of sequences which could serve as target sites, i.e. which have high sequence homologies within the predetermined group of organisms and low homologies for organisms not being members of the groups. For this purpose, sequence alignments were developed, and a number of nucleic acids were designed based on these analyses.
  • Table 1 SEQ ID No. Sequence 5' - 3' SEO ID No. 1 CGTGAGACAGTTCGGTCT SEQ ID No. 2 GACGAACCTCTGGTATGTC SEQ ID No. 3 GGCTGGTTGGCTACGTA SEQ ID No. 4 GGCTGGTTGGCTATGTG SEQ ID No. 5 GATTAGCTACGTTCGGGAT SEQ ID No. 6 ATTAGCTACGTTCGGAAGAG SEQ ID No. 7 ATTAGCTACGTTCGGAAGTG SEQ ID No. 8 GGCTACGTTCGGGAGA SEQ ID No. 9 TCGCTACGTTCGGAAGA SEQ ID No.
  • GTTTTGGTCTATTAGTACCAGTAG SEQ ID No. 69 GTGGTCAATTAGTACCAGTAGC SEQ ID No. 70 GTTGGTGTATTAGTACCAGTCAC SEQ ID No. 71 TCGGTAAATTAGTACCAGTCAC SEQ ID No. 72 GGCCAATTAGTACCAGTCAC SEQ ID No. 73 GGTCAATTAGTACCGGTCAC SEQ ID No. 74 GGCTAATTAGTACCGGTCAC SEQ ID No. 75 GGTGTATTAGTACCGGTCG SEQ ID No. 76 AAGTCATCGGCCTATTAGTACT SEQ ID No. 77 AAGTCTTCGGTGTATTAGTACCA SEQ ID No.
  • Table 2 "Micrococcus" Group of Organisms including the genera Micrococcus, Kocuria, Kytococcus, Nesterenkonia (all formerly classified as Micrococcus ) SEQ IDs preferred primers 2, 7, 9 / 64, 79, 81, 86, 87 preferred probe pairs 10 - 12 / 36 - 40 Micrococcus Genus SEQ IDs preferred primers 2 / 86 further possible primers 5 or 27 or 28 or 51 / 63 or 76 preferred probe pairs 10, 11 / 39 further possible probe pairs 30 / 13 59, 61 / 56 35 / 41 probe pair for identification of Micrococcus luteus 55 / 58 Kocuria species SEQ IDs Kocuria rosea preferred primers 9 / 81 Kocuria rosea preferred probe pairs 10 / 38 Kocuria rosea further possible probe pairs 33 / 13 65 / 62 35 / 43 Kocuria kristinae
  • a nucleic acid is a DNA, RNA, PNA, or LNA which is obtained either through isolation from genomic DNA or from cDNA according to known standard methods and purified or generated artificially using known methods such as oligonucleotide synthesis or isolated as ribosomal RNA or mRNA from the organism or synthesised as PNA or LNA.
  • a “PNA” is a peptide nucleic acid in which instead of the phosphoric acid backbone of the DNA, 2-aminoethylglycin compounds occur.
  • a “LNA” is a locked nucleic acid in which the pentose moiety of DNA or RNA is modified to contain a 2'-O, 4'-C methylene linkage (1,2:5,6-di-O-isopropylene-alpha-D-allofuranose) to "lock" the nucleic acid in a certain conformational state.
  • nucleic acids in the nucleic acids, up to 20 % of the nucleotides in 10 consecutive nucleotides, preferably 1 nucleotide from a block of 10 consecutive nucleotides may be replaced by nucleotides (e.g . inosin, etc.) which do not naturally occur in bacteria.
  • nucleotides e.g . inosin, etc.
  • the nucleic acids may further contain modifications which allow the production of a signal that can be detected directly or indirectly.
  • the expert is aware of the following modifications here:
  • Fragments of oligonucleotides arise due to deletion of one or more nucleotides on the 5' and/or 3' end of an oligonucleotide.
  • partial sequences of a larger polynucleotide are considered. These partial sequences comprise ten nucleotides and are then identical when all 10 modules are identical for two comparative sequences. The nucleotides thymidine and uridine are identical. As partial sequences, all possible fragments of a larger polynucleotide can be considered.
  • polynucleotides which comprise 20 nucleotides and which differ in the 5th module.
  • sequence comparison six 10-way nucleotides are found which are identical and five which are not identical, because they differ in one module.
  • the identity can be gradually determined, whereby the unit is stated in percent.
  • the degree of identity partial sequences are also considered, which comprise as a minimum the length of the actually used sequence, e.g. as primer, or 20 nucleotides.
  • polynucleotide A with a length of 100 nucleotides and B with a length of 200 nucleotides are compared.
  • a primer with a length of 14 nucleotides is derived from polynucleotide B.
  • polynucleotide A is compared with the primer over its complete length. If the sequence of the primer occurs in polynucleotide A, whereby it however deviates in one module, then there is a fragment with a degree of identity of 13:14, i.e . approx. 93%.
  • polynucleotides A and B previously mentioned are compared in their entirety. In this case all the possible comparative windows of a length of 20 nucleotides are applied and the degree of identity determined for them. If then nucleotides nos. 50-69 of polynucleotide A and B are identical with the exception of nucleotide no. 55, then a degree of identity of 19:20, i.e. 95% arises for these fragments.
  • Primers are oligonucleotides which act as starter molecules during a PCR. Here, they hybridize on a target molecule, which may be, for example, DNA or RNA, and are lengthened by a polymerase. They can also however act as probes.
  • Probes are oligonucleotides or polynucleotides which hybridize on the target nucleic acid molecules. They are used for the direct or indirect detection of these target nucleic acid molecules. For this purpose, they can be coupled to fluorescent molecules or to molecules containing colouring agents. In addition they can be indirectly detected with an ELISA (enzyme-linked immunosorbent assay). In a special version they only produce a signal through FRET (Fluorescence Resonance Energy Transfer) when two probes hybridize adjacently in a defined manner. In this case a colouring agent on a probe is excited by a light beam and transfers its excitation energy to the colouring agent of the adjacent probe. This then emits light of a defined wavelength. They can also be used as primers.
  • FRET Fluorescence Resonance Energy Transfer
  • a multiplex PCR is a Polymerase Chain Reaction or nucleic acid amplification reaction in which more than two primers are used which are not regarded as a forwards-backwards primer pair. With the presence of all nucleotide target molecules to be detected, this leads to the creation of at least two different amplicons. These amplicons should at least differ in the region in which the primers link, but they can also be allocated to completely different genes.
  • real-time detection is defined as the simultaneous running of two processes: the detection of the DNA or RNA and a process which leads to the provision of a detectable amount of DNA or RNA.
  • the release of genomic DNA/RNA from cells may, for example, be involved or the enrichment of DNA/RNA from a complex mixture or the amplification of polynucleotides, e.g . through a PCR.
  • Detection is the perception of a signal which correlates to the presence and possibly the amount of the DNA/RNA. In the case of the PCR this type of signal may increase with the increasing amplification of the target DNA.
  • Real-time detection can be carried out also in a miniaturised form, e.g . on a chip.
  • the signal can, for example, be produced through the fluorescent molecules of a probe, through radioactive molecules or through enzyme-coupled colour or fluorescence intensity.
  • real-time detection is synonymous to on-line detection.
  • Derivatives or variants of the oligonucleotides according to the invention are taken to mean sequences which differ in at least one nucleotide from the specific sequences according to SEQ ID numbers 1-108, for example, by at least one base interchange, an insertion, deletion or addition.
  • These also include oligonucleotides which are at least 80 % identical to one of the specific sequences according to SEQ ID numbers 1-108 and oligonucleotides with a comparable specificity of hybridization. The latter means that the derivative produces the same hybridization pattern with a specified sample containing nucleic acid, such as the oligonucleotide with one of the specific sequences with one of the SEQ ID numbers 1-108.
  • variants of any nucleic acid molecule of any of SEQ ID NO 1-108 are contemplated as alternatives to the respective oligonucleotide of SEQ ID NO 1-108.
  • a variant of a nucleic acid molecule of any of SEQ ID NO 1-108 is
  • Biochip is taken to mean carriers for the high throughput of analyses as marketed, for example, by AFFYMETRIX.
  • the chips enable the testing of numerous different nucleic acids on one carrier.
  • hybridization is understood as the double strand formation of two identical or similar nucleic acid fragments (DNA, RNA, PNA, LNA). Specific hybridization is the term used if the hybridization is carried out under stringent conditions and gives a stable hybrid nucleic acid.
  • sequence which specifically hybridizes with a sequence according to (i) refers to a sequence which hybridizes under stringent conditions with the sequence according to (i).
  • the stringency of the hybridization conditions depends on various factors such as the base composition of the hybrid, the level and geometry of mispairing between the two nucleic acids, as well as reaction parameters like ionic strength and the reaction temperature. Appropriate conditions can be determined based on thermodynamic calculations (SantaLucia et. al. 1996), which have been incorporated into a variety of computer programs known to those skilled in the art. Examples of stringent conditions are set forth in the example section below.
  • a melting curve analysis of a sample is defined as the following sequence of processes: the heating of the sample to a temperature high enough to denature the nucleic acids in the sample; the cooling of the sample to a temperature below the annealing temperature of the target nucleic acids; a slow heating of the sample with the simultaneous (real-time, online) detection of the dissociation (i.e., melting) of the nucleic acids.
  • Detection is the perception of a signal which correlates to the proportion of dissociated and not yet dissociated nucleic acids. In the case of a melting curve after a PCR with hybridization probes or with a double strand binding dye, this type of signal may decrease with the increasing temperature.
  • An inflexion point of the signal course is defined as melting temperature.
  • the melting temperature of a sample is the point at which half the probes or the double strand binding dye have melted off the target nucleic acids.
  • the first negative derivative of the signal course can be plotted against the temperature; the melting temperatures then appear as peaks of the curve.
  • a melting curve can also be carried out in a miniaturised form, e.g. on a chip.
  • the nucleic acid to be examined is passed to a PCR.
  • a PCR This has the result that specific amplicons are produced if nucleic acids of bacteria belonging to the baseline flora in environmental monitoring are present in the sample.
  • the PCR can be arranged as a simple linear PCR with only one oligonucleotide as primer, but preferably the PCR takes place however with so-called forwards and backwards primers for each genome section of the bacterial nucleic acid to be amplified.
  • oligonucleotides and therefore various PCR runs are carried out in the form of a multiplex PCR.
  • different amplicons are created in the PCR in a single initiated reaction with the aid of the various oligonucleotides.
  • oligonucleotides of this invention specific for the genera Micrococcus, Kocuria, Kytococcus, Nesterenkonia, and Corynebacterium with oligonucleotides for the detection of the genus Staphylococcus.
  • biochips chip technology
  • the individual spots on the chip contain analysis material from different sources.
  • the chip can carry a set of oligonucleotides, whereby each spot contains a specific oligonucleotide and this oligonucleotide pattern is brought into contact with analysis samples.
  • the analysis material contains nucleic acids of bacteria belonging to the baseline flora in environmental monitoring, it hybridizes with the specific probes present on the chip and produces a corresponding signal pattern.
  • the detection method can include further steps, such as for example an amplification of the nucleic acid to be detected, whereby this preferably occurs using PCR and/or a southern hybridization with specific probes, whereby this hybridization occurs without prior amplification or after amplification of the nucleic acid to be detected is concluded.
  • the nucleic acid to be detected can be detected using the ligase chain reaction.
  • the nucleic acid to be detected can be enriched by isothermal nucleic acid amplification.
  • the amplification of the target nucleic acid can also take place using real-time detection.
  • the amplification of the nucleic acid to be detected and/or the detection of the contained amplicons occurs on a biochip, whereby it is particularly preferable to carry out the amplification and detection on one chip.
  • oligonucleotides are selected from a nucleic acid, comprising at least one sequence with one of the SEQ ID numbers 1-108 or derivatives thereof.
  • the stated oligonucleotides can on one hand be used as primers within the scope of a PCR and on the other hand also as probes, for example within the scope of a Southern blot hybridization.
  • the specialist can form the suitable combination of oligonucleotides as primers or probes.
  • the stated oligonucleotides or combinations of them are used in the form of a kit for the classification of environmental organisms, whereby the kit also includes other reagents for the detection of bacteria or for conducting the detection reactions.
  • the kit also includes other reagents for the detection of bacteria or for conducting the detection reactions.
  • the reagents and enzymes required for the PCR and, where applicable, suitable carrier materials are also included, for example, such as is desired with the chip technology.
  • oligonucleotides or oligonucleotide combinations according to the invention are therefore a suitable means for the specific identification of bacteria belonging to the baseline flora in environmental monitoring.
  • an amplification control nucleic acid is added during amplification. Accordingly, not only the microbial nucleic acid or a part thereof is amplified, but also the amplification control nucleic acid, which means that at least one amplification fragment in each case is created.
  • the amplification control nucleic acid e.g . a DNA fragment, is here an "internal standard molecule" which serves as an indicator for the effectiveness of the reaction (Ballagi-Pordany, Belak 1996). It is added in a defined quantity to the amplification reaction and amplified in parallel.
  • the amplification control nucleic acid is preferably single or double stranded and may be of unlimited length. Amplification control nucleic acids with a length of up to a thousand nucleotides have proven successful.
  • Genomic DNA of single colonies from agar plates was isolated using known standard methods. The DNA samples were then added to six PCR mixes with different primer pairs with the following composition (final volume 20 ⁇ l): Component Final Concentration H 2 O - PCR buffer, 10 x conc. 1 x conc. MgCl 2 4 mM dATP, dCTP, dGTP, dUTP 200 ⁇ M each Primer forward (see below) 400 nM Primer reverse (see below) 400 nM Taq DNA Polymerase 0.125 U/ ⁇ l SYBR Green I 0.25 x conc. Sample DNA var. Mix 1 Mix 2 Mix 3 Mix 4 Mix 5 Mix 6 Primer forward SEQ ID No. 5 SEQ ID No. 6 SEQ ID No. 5 SEQ ID No. 9 SEQ ID No. 8 SEQ ID No. 8 Primer reverse SEQ ID No. 64 SEQ ID No. 64 SEQ ID No. 76 SEQ ID No. 81 SEQ ID No. 87 SEQ ID No. 83
  • the PCR was carried out on a LightCycle ® 1.1 instrument (Roche Diagnostics) using glass capillaries and the following time/temperature protocol: Initial Denaturation 95°C 2 min Amplification (35 cycles) 95°C 2 s 62°C 20 s 72°C 10 s (fluorescence acquisition) Melting Curve 95°C 0 s 40°C 45 s 95°C 0 s (continuous fluorescence acquisition) Cooling 40°C 30 s
  • Table 3 PCR results of Micrococcus and Kocuria isolates with different primer pairs.
  • Mix 4 Mix 5
  • Each of the primer combinations specifically detected only one of the tested species.
  • Genomic DNA of single colonies from agar plates was isolated using known standard methods. The DNA samples as well as an internal amplification control DNA were then added to the PCR mix with the following composition (final volume 20 ⁇ l): Component Final Concentration H 2 O - PCR buffer, 10 x conc. 1 x conc. MgCl 2 4 mM dATP, dCTP, dGTP, dUTP 200 ⁇ M each SEQ ID No. 5, 6, 8, 9 200 nM each SEQ ID No. 7 100 nM SEQ ID No. 64, 81, 86, 87, 92 250 nM each SEQ ID No. 30 - 34 a) 50 nM each SEQ ID No. 13 b) 250 nM SEQ ID No.
  • the PCR was carried out on a LightCycle ® 2.0 instrument (Roche Diagnostics) using glass capillaries and the following time/temperature protocol: Initial Denaturation 95°C 2 min Amplification (35 cycles) 95°C 2 s 62°C 20 s (fluorescence acquisition) 72°C 10 s Melting Curve 95°C 0 s 40°C 45 s 80°C 0 s (continuous fluorescence acquisition) Cooling 40°C 30 s
  • the software modules "Qualitative Detection” and “Tm Analysis” of the LightCycler ® 2.0 instrument software were used to analyze amplification and melting curves, respectively.
  • Amplification of DNA of Staphylococcus spp. was detected in channel 640.
  • the internal amplification control was detected in channel 705 for all samples that were negative in channels 640 and 670.
  • the suitability of these samples for PCR was shown with a consensus PCR system targeting the 16S rDNA of eubacteria (Lane 1991).
  • Table 4 shows a summary of the results.
  • Table 4 Results of the comparative study, grouped by PCR results. result conventional methods PCR result, confirmed by sequencing correct incorrect total negative - neither Staphylococcus nor Micrococcus / Kocuria / Kytococcus 62 7 69 positive for Staphylococcus 15 7 22 positive for Micrococcus luteus 28 12 40 positive for Micrococcus / Kocuria / Kytococcus (without M . luteus) 8 11 19 Total 113 37 150
  • Genomic DNA of single colonies from agar plates was isolated using known standard methods. The DNA samples were then added to the PCR mix with the following composition (final volume 20 ⁇ l): Component Final Concentration H 2 O - PCR buffer, 10 x conc. 1 x conc. MgCl 2 4 mM dATP, dCTP, dGTP, dUTP 200 ⁇ M each SEQ ID No. 1 750 nM SEQ ID No. 90 400 nM SEQ ID No. 47 - 49 a) 100 nM each SEQ ID No. 25 b) 200 nM Taq DNA Polymerase 0.1 U/ ⁇ l Sample DNA var. a) 3'-labeled with Fluorescein b) 5'-labeled with LC-Red 610 and 3'-blocked with a phosphate moiety
  • the PCR was carried out on a LightCycle ® 2.0 instrument (Roche Diagnostics) using glass capillaries and the following time/temperature protocol: Initial Denaturation 95°C 2 min Amplification (35 cycles) 95°C 2 s 62°C 20 s (fluorescence acquisition) 72°C 10 s Melting Curve 95°C 0 s 40°C 45 s 80°C 0 s (continuous fluorescence acquisition) Cooling 40°C 30 s
  • Amplification of DNA of Corynebacterium spp. was detected in channel 610.
  • Table 5 lists the tested species and the respective PCR result. Table 5: Tested isolates and PCR results. Species PCR Result Actinomyces naeslundii - Brevibacterium casei - Brevibacterium sanguinis - Corynebacterium afermentans + Corynebacterium amycolatum + Corynebacterium aurimucosum + Corynebacterium coyleae + Corynebacterium genitalium + Corynebacterium glutamicum + Corynebacterium jeikeium + Corynebacterium kroppenstedtii + Corynebacterium macginleyi + Corynebacterium minutissimum + Corynebacterium mucifaciens + Corynebacterium simulans + Corynebacterium suicordis + Corynebacterium thomssenii + Corynebacterium tuberculostearicum + Exiguobacterium sp.
  • Genomic DNA of single colonies from agar plates was isolated using known standard methods. The DNA samples as well as an internal amplification control DNA were then added to the PCR mix with the following composition (final volume 20 ⁇ l): Component Final Concentration H 2 O - PCR bluffer, 10 x conc. 1 x conc. MgCl 2 4mM dATP, dCTP, dGTP, dUTP 200 ⁇ M each SEQ ID No: 1, 2, 7, 9 250 nM each SEQ ID No: 67, 68, 69, 70, 71, 73, 75 600 nM each SEQ ID No: 64, 79, 81, 86, 87 500 nM each SEQ ID No: 10.
  • the PCR was carried out on a LightCycler ® 2.0 instrument (Roche Diagnostics) using glass capillaries and the following time/temperature protocol: Initial Denaturation 95°C 2 min Amplification (35 cycles) 95°C 2 s 62°C 20 s (fluorescence acquisition) 72°C 10 s Melting Curve 95°C 0 s 40°C 45 s 80°C 0 s (continuous fluorescence acquisition) Cooling 40°C 30 s
  • the software modules "Qualitative Detection” and “Tm Analysis” of the LightCycle ® 2.0 instrument software (Roche Diagnostics) were used to analyze amplification and melting curves, respectively.
  • Amplification of DNA of Corynebacterium spp. was detected in channel 610, and a melting curve analysis was performed in channel 610.
  • Amplification of DNA of Staphylococcus spp. and Macrococcus spp. was detected in channel 640, and differentiation of the two genera was performed via melting curve analysis in channel 640.
  • Corynebacterium All isolates that belonged to the genus Corynebacterium were detected in channel 610, no false negatives occurred. They all had a melting peak or shoulder at temperatures higher than 63°C in channel 610. Some coryneform bacteria had a melting curve in channel 610 as well, but with all peaks occurring at temperatures below 63°C.
  • the second melting group comprised different Micrococcus spp. (but not M. luteus ) and Kocuria palustris .
  • the melting peak temperatures ranged from approx. 52°C for two isolates that phylogenetically belong to the new species Micrococcus chenggongense up to 56°C for Kocuria palustris.
  • the internal amplification control was detected in channel 705 for all samples that were negative in channels 610, 640, and 670. The suitability of these samples for PCR was shown with a consensus PCR system targeting the 16S rDNA of eubacteria (Lane 1991).
  • Table 6 Isolates of the genus Corynebacterium: positive amplification signal in channel 610, at least one melting peak or shoulder at temperatures above 63°C in melting curve channel 610.
  • Corynebacterium afermentans 9 Corynebacterium amycolatum 1 Corynebacterium appendicis 2 Corynebacterium aurimucosum 1 Corynebacterium caspium 1 Corynebacterium coyleae 4 Corynebacterium durum 1 Corynebacterium genitalium 1 Corynebacterium glutamicum 1 Corynebacterium imitans 1 Corynebacterium jeikeium 2 Corynebacterium kroppenstedtii 1 Corynebacterium kutscheri 1 Corynebacterium lipophiloflavum 1 Corynebacterium macginleyi 2 Corynebacterium minutissimum 3 Corynebacterium mucifaciens 5 Corynebacterium phocae 1 Corynebacterium riegelii 1 Corynebacterium simulans 1 Corynebacterium singulare 2 Corynebacterium suicord
  • Isolates of the genus Staphylococcus positive amplification signal in channel 640, at least one melting peak or shoulder at temperatures 64°C in melting curve channel 640; characteristic melting peak temperatures in channel 705 are given in the last column of the table.
  • Positive amplification signal in channel 705 (Internal Control). Species Taxonomic Group Number of Isolates Achromobacter xylosoxidans Betaproteobacteria 1 Actinomyces bovis Actinomycetales 1 Actinomyces sp.

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Claims (11)

  1. Kit, umfassend eine Kombination von Nucleinsäuremolekülen, die beim Nachweis der taxonomischen Einheit, umfassend die Gattungen Micrococcus, Kocuria, Kytococcus, Nesterenkonia oder daraus bestehend, geeignet sind, wobei die Kombination umfasst
    (i)
    (a) die Kombination der SEQ ID NO: 2, 7 und 9;
    (b) die Kombination eines Nucleinsäuremoleküls, das unter stringenten Bedingungen mit SEQ ID NO: 2 hybridisiert, eines Nucleinsäuremoleküls, das unter stringenten Bedingungen mit SEQ ID NO: 7 hybridisiert, und eines Nucleinsäuremoleküls, das unter stringenten Bedingungen mit SEQ ID NO: 9 hybridisiert;
    (c) die Kombination eines Nucleinsäuremoleküls, das zu mindestens 90% mit SEQ ID NO: 2 identisch ist, eines Nucleinsäuremoleküls, das zu mindestens 90% mit SEQ ID NO: 7 identisch ist, eines Nucleinsäuremoleküls, das zu mindestens 90% mit SEQ ID NO: 9 identisch ist, oder
    (d) die Kombination der Komplemente der Nucleinsäuremoleküle nach (i) (a), (b) oder (c); zusammen mit
    (ii)
    (a) der Kombination der SEQ ID NO: 64, 79, 81, 86 und 87;
    (b) der Kombination eines Nucleinsäuremoleküls, das unter stringenten Bedingungen mit SEQ ID NO: 64 hybridisiert, eines Nucleinsäuremoleküls, das unter stringenten Bedingungen mit SEQ ID NO: 79 hybridisiert, eines Nucleinsäuremoleküls, das unter stringenten Bedingungen mit SEQ ID NO: 81 hybridisiert, eines Nucleinsäuremoleküls, das unter stringenten Bedingungen mit SEQ ID NO: 86 hybridisiert, und eines Nucleinsäuremoleküls, das unter stringenten Bedingungen mit SEQ ID NO: 87 hybridisiert;
    (c) der Kombination eines Nucleinsäuremoleküls, das zu mindestens 90% mit SEQ ID NO: 64 identisch ist, eines Nucleinsäuremoleküls, das zu mindestens 90% mit SEQ ID NO: 79 identisch ist, eines Nucleinsäuremoleküls, das zu mindestens 90% mit SEQ ID NO: 81 identisch ist, eines Nucleinsäuremoleküls, das zu mindestens 90% mit SEQ ID NO: 86 identisch ist, und eines Nucleinsäuremoleküls, das zu mindestens 90% mit SEQ ID NO: 87 identisch ist, oder
    (d) der Kombination der Komplemente der Nucleinsäuremoleküle nach (ii) (a), (b) oder (c).
  2. Kit nach Anspruch 1, ferner umfassend eine Nucleinsäure, ausgewählt aus
    (a) SEQ ID NO: 10, 11, 12, 36, 37, 38, 39 oder 40;
    (b) einem Nucleinsäuremolekül, das unter stringenten Bedingungen mit einem Nucleinsäuremolekül nach (a) hybridisiert;
    (c) einem Nucleinsäuremolekül, das zu mindestens 90% mit dem Nucleinsäuremolekül nach (a) identisch ist, oder
    (d) dem Komplement eines Nucleinsäuremoleküls nach (a) bis (c).
  3. Kit nach einem der Ansprüche 1 bis 2, der auch zur Verwendung beim Nachweis der Gattung Corynebacterium geeignet ist, wobei die Kombination ferner umfasst
    i) SEQ ID NO: 1 oder eine Variante davon und
    eine aus SEQ ID NO: 67, 68, 69, 70, 71, 73, 75 oder eine jeweilige Variante von einer von diesen oder
    ii) SEQ ID NO: 3 und 4 oder eine jeweilige Variante von einer von diesen und eine aus den SEQ ID NO: 46, 90 oder 91 oder eine jeweilige Variante von einer von diesen,
    wobei es sich bei einer Variante eines Nucleinsäuremoleküls nach i) oder ii)
    a) um ein Nucleinsäuremolekül handelt, das unter stringenten Bedingungen mit dem Nucleinsäuremolekül hybridisiert;
    b) um ein Nucleinsäuremolekül handelt, das zu mindestens 90% mit dem Nucleinsäuremolekül nach a) identisch ist, oder
    c) um das Komplement des Nucleinsäuremoleküls oder einer der Varianten a) bis b) handelt.
  4. Kit nach einem der Ansprüche 1 bis 4, der auch zur Verwendung beim Nachweis der Gattung Staphylococcus geeignet ist, wobei die Kombination ferner umfasst
    (i) SEQ ID NO: 93 oder eine Variante davon und
    eine aus SEQ ID NO: 94 - 101 oder eine jeweilige Variante von einer von diesen;
    wobei es sich bei einer Variante eines Nucleinsäuremoleküls nach i)
    a) um ein Nucleinsäuremolekül handelt, das unter stringenten Bedingungen mit dem Nucleinsäuremolekül hybridisiert;
    b) um ein Nucleinsäuremolekül handelt, das zu mindestens 90% mit dem Nucleinsäuremolekül nach a) identisch ist, oder
    c) um das Komplement des Nucleinsäuremoleküls oder einer der Varianten a) bis b) handelt.
  5. Verfahren zum Amplifizieren oder Identifizieren von Bakterien-DNA der taxonomischen Einheit, umfassend die Gattungen Micrococcus, Kocuria, Kytococcus, Nesterenkonia oder daraus bestehend, in einer Probe, wobei
    (a) in einem ersten Amplifikationsschritt die DNA der taxonomischen Einheit mit konservierten Primern amplifiziert wird, wobei die in dem ersten Amplifikationsschritt verwendeten Primer alle in Anspruch 1 (i) (a) definierten Nucleinsäuren oder alle in Anspruch 1 (i) (b) definierten Nucleinsäuren oder alle in Anspruch 1 (i) (c) definierten Nucleinsäuren zusammen mit allen in Anspruch 1 (ii) (a) definierten Nucleinsäuren oder allen in Anspruch 1 (ii) (b) definierten Nucleinsäuren oder allen in Anspruch 1 (ii) (c) definierten Nucleinsäuren umfassen.
  6. Verfahren nach Anspruch 5, wobei
    (b) in einem weiteren Nachweisschritt die durch den Amplifikationsschritt (a) erhaltenen DNA-Fragmente mittels Sonden nachgewiesen werden,
    wobei die in Schritt b) verwendeten Sonden eine oder mehrere der in Anspruch 2 (a) bis (d) definierten Nucleinsäuren umfassen.
  7. Verfahren nach einem der Ansprüche 5 oder 6, das in der gleichen Probe auch das Amplifizieren oder Identifizieren der Gattung Corynebacterium und/oder Staphylococcus ermöglicht, wobei bei dem Verfahren
    i) in einem zweiten Amplifikationsschritt, der gleichzeitig mit dem ersten Amplifikationsschritt oder anschließend stattfindet, die DNA der Gattung Corynebacterium mit konservierten Primern amplifiziert wird, und/oder
    ii) in einem dritten Amplifikationsschritt, der gleichzeitig mit dem ersten Amplifikationsschritt oder anschließend stattfindet, die DNA der Gattung Staphylococcus mit konservierten Primern amplifiziert wird.
  8. Verfahren nach einem der Ansprüche 5 bis 7, wobei die in dem zweiten Amplifikationsschritt verwendeten Primer eine oder mehrere der Nucleinsäuren umfassen, ausgewählt aus
    a) SEQ ID NO: 1, 3 oder 4,
    b) SEQ ID NO: 46, 90, 91, 67, 68, 69, 70, 71, 73, 75, 72 oder 74;
    c) einem Nucleinsäuremolekül, das unter stringenten Bedingungen mit dem Nucleinsäuremolekül nach einem von a) bis b) hybridisiert;
    d) einem Nucleinsäuremolekül, das zu mindestens 90% mit dem Nucleinsäuremolekül nach a) oder b) identisch ist, oder
    e) dem Komplement des Nucleinsäuremoleküls nach einem von a) oder b).
  9. Verfahren nach einem der Ansprüche 5 bis 8, wobei die in dem dritten Amplifikationsschritt verwendeten Primer eine oder mehrere der Nucleinsäuren umfassen, ausgewählt aus
    a) SEQ ID NO: 93;
    b) SEQ ID NO: 94, 95, 96, 97, 98, 99, 100 oder 101;
    c) einem Nucleinsäuremolekül, das unter stringenten Bedingungen mit dem Nucleinsäuremolekül nach einem von a) bis b) hybridisiert;
    d) einem Nucleinsäuremolekül, das zu mindestens 90% mit dem Nucleinsäuremolekül nach a) oder b) identisch ist, oder
    e) dem Komplement des Nucleinsäuremoleküls nach einem von a) oder b).
  10. Verfahren nach einem der Ansprüche 5 bis 9, das ferner die Amplifikation oder den Nachweis von einer oder mehreren der Gattungen Micrococcus, Kocuria, Kytococcus und/oder Nesterenkonia oder von einer oder mehreren Spezies innerhalb einer oder mehrerer dieser Gattungen ermöglicht.
  11. Verfahren nach einem der Ansprüche 5 bis 10, das ferner die Amplifikation oder den Nachweis von einer oder mehreren aus Micrococcus luteus, Staphylococcus aureus oder Dermacoccus nishinomiyaensis ermöglicht.
EP09010238.5A 2009-08-07 2009-08-07 Nukleinsäuren und Verfahren zur Umweltüberwachung Active EP2287332B1 (de)

Priority Applications (2)

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PCT/EP2010/004793 WO2011015355A2 (en) 2009-08-07 2010-08-04 Nucleic acids and methods for environmental monitoring

Applications Claiming Priority (1)

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DE19515891C2 (de) * 1995-04-29 1999-10-28 Roche Diagnostics Gmbh Gattungs- und speziesspezifische Identifizierung von Legionellen
DE19731292A1 (de) * 1997-07-21 1999-01-28 Biotecon Ges Fuer Biotechnologische Entwicklung & Consulting Mbh Nucleinsäuremolekül, Kit und Verwendung
DE19739611A1 (de) * 1997-09-09 1999-03-11 Biotecon Ges Fuer Biotechnologische Entwicklung & Consulting Mbh Nucleinsäure-Sequenzen und Verfahren zum Nachweis von Bakterien der Gattung Pseudomonas
DE19945916A1 (de) * 1999-09-24 2001-04-05 Biotecon Diagnostics Gmbh Nukleinsäuremoleküle zum Nachweis von Bakterien und phylogenetischen Einheiten von Bakterien

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